scholarly journals Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

2005 ◽  
Vol 71 (9) ◽  
pp. 5624-5626 ◽  
Author(s):  
X. C. Shan ◽  
P. Wolffs ◽  
M. W. Griffiths
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Quantitative Detection ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Immunomagnetic Capture ◽  
Capture Method

ABSTRACT In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold.

scholarly journals New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance

2019 ◽  
Vol 57 (9) ◽  
Author(s):  
William S. Probert ◽  
Jill K. Hacker
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Outbreak Detection ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Laboratory Surveillance

ABSTRACTLaboratory surveillance plays an important role in the detection and control of hepatitis A outbreaks and requires the application of rapid and accurate molecular diagnostic tools for hepatitis A virus (HAV) RNA detection, subgenotype identification, and sequence-based genotyping. We describe the development and validation of a triplex real-time, reverse transcription-PCR (triplex rRT-PCR) assay for the identification and discrimination of HAV subgenotypes IA, IB, and IIIA and a singleplex rRT-PCR assay designed to detect all HAV genotypes infecting humans. Overall, the accuracy, sensitivity, and specificity of the new assays were >97% for serum and plasma specimens collected during unrelated outbreaks of HAV in California and Michigan compared to a nested RT-PCR genotyping assay and the ISO 15216-1 rRT-PCR method for HAV detection. The new assays will permit the rapid detection of HAV RNA and discrimination among subgenotypes IA, IB, and IIIA in serum and plasma specimens, which will strengthen public health surveillance efforts for HAV outbreak detection and response.

scholarly journals Development, Evaluation, and Standardization of a Real-Time TaqMan Reverse Transcription-PCR Assay for Quantification of Hepatitis A Virus in Clinical and Shellfish Samples

2006 ◽  
Vol 72 (6) ◽  
pp. 3846-3855 ◽  
Author(s):  
M. Isabel Costafreda ◽  
Albert Bosch ◽  
Rosa M. Pint�
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Accurate Estimation ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Development Evaluation

ABSTRACT A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5′ noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.

scholarly journals Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

2004 ◽  
Vol 70 (7) ◽  
pp. 4371-4374 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real

ABSTRACT In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

scholarly journals Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a fourplex real-time quantitative reverse transcription-PCR assays

2021 ◽  
Author(s):  
Mathieu Durand ◽  
Philippe Thibault ◽  
Simon Lévesque ◽  
Ariane Brault ◽  
Alex Carignan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Internal Control ◽  
Reverse Transcription ◽  
Selective Advantage ◽  
Locked Nucleic Acid ◽  
Quantitative Detection ◽  
Respiratory Pathogens ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

AbstractThe early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of the coronavirus disease 2019 (COVID-19). In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give SARS-CoV-2 a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected ≤ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of 9 logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to detect multiple viral target sequences containing typical mutations of SARS-CoV-2 variants in a single reaction, allowing quicker detection of circulating relevant variants.

scholarly journals Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays

2022 ◽  
Vol 9 (1) ◽  
pp. 1-20
Author(s):  
Mathieu Durand ◽  
Philippe Thibault ◽  
Simon Lévesque ◽  
Ariane Brault ◽  
Alex Carignan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Internal Control ◽  
Reverse Transcription ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Respiratory Pathogens ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected £ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

scholarly journals Implementation of an in-house real-time reverse transcription-PCR assay to detect the emerging SARS-CoV-2 N501Y variants

2021 ◽  
pp. 104868
Author(s):  
Marielle BEDOTTO ◽  
Pierre-Edouard FOURNIER ◽  
Linda HOUHAMDI ◽  
Philippe COLSON ◽  
Didier RAOULT
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Pcr Assay ◽  
Reverse Transcription Pcr
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